Note: Descriptions are shown in the official language in which they were submitted.
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IDENTIFICATION OF FACTORS WHICH MEDIATE THE INTERACTION OF
HETEROTRIMERIC G PROTEINS AND MONOMERIC G PROTEINS
BACKGROUND OF THE INVENTION
Signal transduction pathways linking extracellular factors to the activation
of the
Rho GTPase have been implicated in cell growth control and cytoskeletal
rearrangements.
Specifically, heterotrimeric G proteins have been shoawn to mediate these
pathways,
although the mechanism of mediation has been unclear. The identification of
factors which
interact with both heterotrimeric G proteins and- Rho GTPase would provid., an
important
tool for investigating and controlling various cell processes, including cell
proliferative
diseases.
SUMMARY OF THE INVENTION
The invention relates to a polypeptide, and corresponding nucleic acid.
comprising
an amino acid sequence of a novel RGS domain, obtainable, e.g., from a guanine
nucleotide
exchange factor (GEF ~ protein, where the polypeptide preferably files not
incl~cie a dbl
homology (DH) domain or a pleckstrin homology (PH) domain. Ln a preferred
embodiment,
the polypeptide has GTPase activating activity and binding affinity for an a G
protein
subunit such as Ga.
The polypeptides and nucleic acids can be used as tools for research,
therapeutics,
and diagnostics as discussed below.
The invention also relates to a method of identifying or assaying for a
molecule, or
mixture of molecules, that regulate the binding of an RGS domain of a GEF
protein to a
substrate, e.g., a G protein subunit such as Ga . In one embodiment, the
method involves
incubating, under effective conditions, a polypeptide having an RGS domain of
a GEF
polygeptide, and optionally having GEF activity, with a Ga subunit, or a
fragment thereof,
in the presence and/or absence of a test molecule; and determining whether the
presence of
the test molecule regulates the binding between the polypeptide and the
subunit, or fragment
thereof. As discussed later, various RGS-GEF polypeptides binding substrates
can be
utilized.
In addition, the invention relates to a method of identifying or assaying for
a
molecule, or mixture of molecules, that regulates a stimulatory effect of a
polypeptide
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comprising an RGS domain of a GEF protein on a polypeptide having a GTPase
activity. In
a preferred embodiment, the method comprises incubating a Ga subunit and a GEF
protein,
under effective conditions, in the presence and absence of a test molecule and
determining
whether the presence of the test molecule regulates the stimulatory effect of
the GEF protein
on Ga subunit GTPase activity.
The invention also relates to a method of identifying or assaying for a
molecule that
specifically regulates a stimulatory effect of a first polypeptide, such as an
activated Ga
subunit, or polypeptide having GTPase activity, on a nucleotide exchange
factor activity of a
second polypeptide. The second polypeptide preferably comprises a RGS-GEF
domain
obtainable from a GEF, and more preferably is a guanine nucleotide exchange
factor (GEF)
for a monomeric G protein. In one embodiment of the method, a first assay is
conducted by
incubating an activated Ga subunit with a GEF protein and a monomeric G
protein in the
presence and absence of a test molecule; a second assay is conducted: by
incubating a GEF
protein and a monomeric G protein in the presence and absence of the test
molecule, and a
determination is made as to whether the molecule has a different effect when
the first assay
is compared to the second assay.
The invention further relates to a method of identifying or assaying for a
molecule,
or mixture of molecules, that mimics the stimulatory effect of an activated Ga
subunit on
GEF mediated nucleotide exchange of a monomeric G protein. In one example,
such a
method comprises identifying a test compound that exhibits a binding affinity
for an RGS
domain of GEF proteins, incubating a GEF protein and monomeric G protein in
the
presence or absence of the test compound, determining whether the test
compound exhibits a
stimulatory effect on GEF mediated nucleotide exchange of a monomeric G
protein.
The invention further relates to a method of identifying or assaying for a
molecule,
or mixture of molecules, that mimics the stimulatory effect of an RGS domain
of GEF
polypeptide on Ga subunit GTPase activity. In one example, such a method
comprises
identifying a test compound that exhibits a binding affinity for a Ga subunit
and incubating
a GTP loaded Ga subunit in the presence or absence of the test compound to
determine
whether the test compound exhibits a stimulatory effect on GEF mediated
nucleotide
exchange of a monomeric G protein.
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BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1, Panel A depicts the alignment of the sequences from RGS proteins and
the
N-terminal region of p 115 Rho GEF as performed by Clustal W with a secondary
structure
mask of RGS4 to assign penalties for gaps. The RGS homologous sequences of
Lsc,
KIAA380, and DrhoGEF2 were further added to this alignment by Clustal W and
manual
adjustments. The (a) symbols above RGS4 indicate the a helices of the RGS
domain of
RGS4. Dark shaded boxes indicate conserved residues of the hydrophobic core of
the RGS
structure. Lightly shaded boxes show other conserved residues. Asterisks mark
the residues
of RGS4 which contact Ga;,. Primary sequences used in the alignment are the
following:
rat RGS4 (SwissProt accession number P49799), mouse RGS2 (008849), human GAIP
(P49795), rat RGS12 (008774), rat RGS14 (008773), human p115 (1654344), mouse
Lsc
( 1389756), human ICIAA380 (2224701 ) and Drosophila DrhoGEF2 (2760368).
Figure 1, Panel B depicts constructs of p115 Rho GEF that were employed in the
studies described herein. Numbers indicate the residues of p115 in each
construct. The
RGS, dbl(DH), and pleckstrin (PH) homology regions are indicated.
GST=glutathione-S-
transferase.
Figure 2, Panel A is a graph showing the hydrolysis of GTP bound to Ga;3 and
Ga,2
at 15°C either with (~o) or without (~o) 10 nM p115 Rho GEF.
Figure 2, Panel B is a graph showing the hydrolysis at 4°C of GTP bound
to Ga,
(~) and Ga,2 (o) and in the presence of various concentrations of pl 15 Rho
GEF. The
initial rates of reaction were plotted as a function of the concentration of p
115 Rho GEF.
Figure 3 is a graph showing the hydrolysis at 15°C of GTP bound to Ga,3
and Ga,z
with either full-length p115 Rho GEF (~), 4Np115 (~), or RGS-pl 15 (~), or
without any
p 115 construct ( ~ ).
Figure 4 is a graph showing the hydrolysis of GTP bound to Ga;i, GaZ, GaQ, and
Gas with 100 nM p115 Rho GEF (0), 100 nM RGS4 (o), or buffer control (o).
Assays
were performed at 4°C for Goc;i and Gas, at 15°C for GaZ, and at
20°C for Gocq.
Figure 5 is a graph showing the selective inhibition of p 115 GAP activity by
the
A1F4- activated forms of Ga subunits. Panel A: P115 (400 nM) was incubated on
ice for 15
minutes with various Ga subunits {400 nM) in the presence of 30 ~M A1C13, 10
mM NaF,
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and 10 mM MgSO~,. The mixture was diluted 20-fold, mixed with 0.3 nM Gai~(GTP)
and
the hydrolysis of bound GTP was measured after incubation at 15°C for 2
minutes. Panel B:
PI 15 (400 nM) was incubated with various concentrations of Gal2(GDP-AIF.>')
(~) or
Ga,3(GDP-AIF4 ) (~) as described for Panel A. The mixture was diluted 20-fold,
mixed
with 1nM Ga,~(GTP) at 4°C and the hydrolysis of bound GTP was assessed
over time. The
initial rate of GTPase of Ga,3 was plotted against the final concentration of
a subunit GDP-
A1F4 . The filled triangle indicates the rate of GTPase of Gala without pl 15.
Figure 6, Panel A is an image of an immunoblot showing the detection of myc-
tagged p 115 Rho GEF expression in COS cells using an anti-myc antibody.
Figure 6, Panel B is an image of an immunoblot showing the detection of a
coimmunoprecipitate of pl 15 Rho GEF and Ga,3 using an anti-myc antibody.
Figure 6, Panel C is an image of an immunoblot showing the detection of the
coimmunoprecipitate of p 115 Rho GEF and Gala using an anti-Ga, ~ antibody.
Figure 6, Panel D is an image of an immunoblot showing the detection of p I 15
Rho
GEF and Gala binding when purified Ga,3 is added to immunoprecipitated pl 15
Rho GEF
when using an anti Gal3 antibody.
Figure 7, Panel A is a graph showing the dissociation of bound GDP from 100 nM
RhoA after 10 minutes in the presence or absence of 100 nm Gal3 or Galz and in
the
presence of various concentrations of p115 Rho GEF as indicated.
Figure 7, Panel B is a graph showing the dissociation of GDP from 100 nM RhoA
after 10 minutes in the presence of 25 nm p 115 Rho GEF and the indicated
concentrations of
Gala or Gale. Unstimulated dissociation of GDP from RhoA is indicated by the
lower
dashed line.
Figure 7, Panel C is a graph showing the dissociation of GDP from 100 nM RhoA
after 10 minutes of incubation with pl 15 Rho GEF and Gal that had been
treated with
AMF, GTP~yS or GDP(3S as indicated.
Figure 7, Panel D is a graph showing the dissociation of of GDP from 100 nM
RhoA after 10 minutes of incubation with pl 15 Rho GEF (25 nM) and various Ga
subunits
( 100 nM) as indicated.
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Figure 8, Panel A is a graph showing the association of 1 nM [32P]GTP to 100
nM
RhoA in the presence of the indicated concentrations of truncated for full-
length p115 Rho
GEF as measured by filtration after 30 minutes at 30°C.
Figure 8, Panel B is a graph showing the dissociation of [;H]-GDP from 100 nM
RhoA after incubation for 10 minutes in the presence or absence of 25 nM p 115
Rho GEF,
20 nM Ga, 3, and 300 nM GST-RGSp 115 as indicated.
Figure 8, Panel C is a graph showing the dissociation of [~H]-GDP from 100 nM
RhoA after incubation for 10 minutes in the presence 25 nM p115 Rho GEF and in
the
presence or absence of 25 nM Ga,3 and the indicated concentrations of Ga,2.
Figure 9, Panel A is an image of an immunoblot showing the detection of myc-
tagged KIAA380 (designated FL147) expression in COS cells using an anti-myc
antibody.
Figure 9, Panel B is an image of an immunoblot showing the detection of a
coimmunoprecipitate of KIAA380 (designated FL147) and Ga,z using an anti-Gain
antibody.
Figure 10 is the a listing of the amino acid sequence for pl 15 Rho GEF.. The
RGS
domain is shown by amino acids 45-170.
Figure 11 is a listing of the nucleic acid sequence for p I 15 Rho GEF. The
RGS
domain is encoded by nucleotides 187-564.
Figure 12 is a listing of the amino acid sequence for KIAA380. The RGS domain
is
shown by amino acids 310-432.
Figure 13 is a listing of the nucleic acid sequence for KIAA380. The RGS
domain
is encoded by nucleotides 1673-2041.
Figure 14 is a listing of the amino acid sequence for Lsc. The RGS domain is
shown
by amino acids 43-168.
Figure 15 is a listing of the nucleic acid sequence for Lsc. The RGS domain is
encoded by nucleotides 218-595.
Figure 16 is a listing of the amino acid sequence for DRhoGEF2. The RGS domain
is shown by amino acids 924-1053
Figure 17 is a listing of the nucleic acid sequence for DRhoGEF2. The RGS
domain
is encoded by nucleotides 3185-3574.
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Figure 18 is a homology alignment of the RGS region of several proteins,
including
GEF proteins with RGS domains (e.g. p115 Rho GEF, Lsc, KIAA380, DrhoGEF). The
alignment was performed using the Clustal method with a PAM250 residue weight
table.
DETAILED DESCRIPTION OF THE INVENTION
G proteins transduce signals from a large number of cell surface heptahelical
receptors to various intracellular effectors. Each heterotrimeric G protein is
composed of a
guanine nucleotide-binding a subunit and a high-affinity dimer of (3 and 'y
subunits. Ga
subunits are commonly classified into four subfamilies (GS, G;, Gq, and G,,)
based on their
amino acid sequence homology and function (A.G. Gilman, An~u. Rev. Biochem,
56> 615
(1987); Y. Kaziro et al., Annu. Rev. Biochem., 60, 349 (1991); Hepler and
Gilman, Trends
Biochem. Sci., 17, 383, (1992)). The G,2 subfamily, consists of two identified
members to
date, G,2 and G,3.
In accordance with the present invention, the identification of proteins
having
activity as both a GTPase activating protein (GAP) for the a subunit of a
heterotrimeric G
protein and activity as a guanine nucleotide exchange factor (GEF) activity
for monomeric G
proteins have been described. Also in accordance with the invention, the first
identification
of a protein having GAP activity for the G~2 subfamily of G proteins has been
described.
Also in accordance with the invention, the ability of an a subunit of a
heterotrimeric G
protein to stimulate GEF mediated guanine nucleotide exchange activity of a
monomeric G
protein has been described. GAP and GEF activity, and methods of screening
thereof, are
described in Berman et al., 1996, Cell 86:445 and Hart et al., 1996, J. Biol.
Chem.,
271:25452.
According to the present invention, the GAP activity of GEF proteins has been
correlated with a novel RGS domain obtainable from a GEF protein. The present
invention
relates to all aspects of such an RGS domain, including all aspects of a Rho
GEF such as
pl I5 Rho-GEF. (U.S. Patent Application No. 08/943,768, herein incorporated by
reference).
A GEF protein modulates cell signaling pathways, both in in vitro and in vivo,
by
modulating the guanine nucleotide exchange activity of a GTPase. According to
the present
invention, a GEF protein which also modulates the GTPase activity of a
heterotrimeric Ga
subunit is described. By way of illustration, p 115 Rho-GEF, which modulates
the guanine
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nucleotide exchange activity of a Rho GTPase, as well as the GTPase activity
of the Ga,z
family of heterotrimeric G protein subunits is described.
The present invention particularly relates to polypeptides comprising a RGS
domain
of a GEF polypeptide, or fragments thereof, and corresponding nucleic acids.
S The invention also relates to methods of using such polypeptides, nucleic
acids, or
derivatives thereof, e.g., in therapeutics, diagnostics, and as research
tools. Other aspects of
the present invention relate to antibodies and other ligands which recognize
the RGS domain
of GEF polypeptides or nucleic acids, methods for identifying or assaying
modulators of the
GEF activities and/or the GAP activities of a protein containing a RGS domain,
and
methods of treating pathological conditions associated with or related to the
RGS domain,
e.g., a GEF mediated interaction of a Ga subunit and a Rho GTPase.
As used herein, an "RGS-GEF polypeptide" means, e.g., a polypeptide containing
an
RGS domain derived from a GEF protein, such as pl 15 Rho-GEF, Lsc, KIAA0380,
or
DRhoGEF2, and, which has one or more of the following activities: a specific
binding
affinity for a polypeptide substrate, e.g., a G protein subunit, preferably an
a subunit, such
as Gi2 or G,3; a GTPase activating activity (GAP), such as a GAP activity for
a G protein a
subunit; or, an immunogenic activity. An RGS-GEF polypeptide preferably does
not
contain a (dbl homology) DH or a (pleckstrin homology) PH domain. DH and PH
domains
are disclosed in Cerione and Zheng, 1996, Curr. Opin. In Cell Biol., 8:216.
For example, the
amino acid sequence of p 115 Rho GEF (Fig.10) contains a novel RGS domain at
amino
acids 45-170, the DH domain at amino acids 420-637, and the PH domain at amino
acids
64.6-672. By "derived," it is meant that the amino acid sequence is obtainable
from a
naturally-occurnng GEF (such as p115, Lsc, KIAA380, and DrrhoGEF2) or a non-
naturally-
occurring "mutated" sequence which is based upon a naturally-occurring GEF
sequence
(i.e., different amino acid residues have been substituted for the amino acid
residues which
occur in the naturally-occurring sequence at a particular position). The
polypeptide can be
"isolated," i.e., the material is in a form in which it is not found in its
original environment,
e.g., more concentrated, more purified, or separated from other
components,etc. A preferred
RGS polypeptide possesses both a GAP and GEF activity, e.g., a mutated pl 15
Rho-GEF.
See below.
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An RGS-GEF nucleic acid codes for an RGS-GEF polypeptide. The nucleic acid
refers to both sense and anti-sense nucleic acids.
By the term "specific binding affinity," it is meant, e.g., that the RGS-GEF
polypeptide has a binding preference for the activated state or transition
state of a G protein
subunit as compared to a GDP-bound state or the nucleotide depleted state. By
"GEF
activity," it is meant, e.g., that the polypeptide stimulates or catalyzes the
dissociation of
GDP from a monomeric G-protein, such as Rho, and subsequent binding of GTP.
Monomeric G-proteins include but are not limited to G-proteins in the Ras,
Rho/Rac, Sar,
Rab, Arf, and Ran families. Of particular interest are the RGS domains of the
following
GEF proteins: human p 115 ( 1654344) (Fig. 10, RGS domain at amino acids 45-
170 ),
mouse Lsc ( 1389756) (Fig. 14, RGS domain at amino acids 43-168), KIAA380
(2224701 )
(Fig. 12, RGS domain at amino acids 310-432) and Drosophila DrhoGEF2 (2760368)
(Fig.
16, RGS domain at amino acids 924-1053).
Another aspect of the invention relates to novel consensus sequences for RGS
domains) of a GEF protein, herein referred to as a "sub-RGS consensus
sequence." An
"RGS domain," as used herein, refers to the amino acid sequence of protein
which is able to
bind to or physically interact with a G protein and, optionally, stimulates
GTPase activity of
that protein. A "sub-RGS consensus sequence," as used herein, refers to a
consensus
sequence which can be used to identify a specific subset of proteins which
contain an RGS
domain. For example, a homology alignment of the RGS domain from several
proteins as
shown and described in Fig. 18 and the corresponding legend, shows that
several sub-RGS
consensus sequences may be defined by the gap of 13 to 14 amino acids that is
apparent in
the RGS domains of GEF proteins. One of these consensus sequences, herein
designated as
"RGS-GEF consensus 1," is herein defined to be a consensus sequence of AA,-AA2-
AA3-
AA4-AAS-AA6-AA7-AA$-(gap of 13 amino acids)-AA22 -AA23-AA24-AA25-AA26,
wherein:
AA, is L;
AA2isEorV;
AA3 is K or P;
AA4 is T, N, or R;
AAS is A;
AA6 is V or P
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AAA is L;
AA8 is either S or a gap of one amino acid, contiguous with the gap of 13
amino acids;
AA22 is either R or W;
AA23 is either V or Y;
AA24 is either P,K, or R
AA~s is either V, I, or Q;
AA26.is either P or D.
A second consensus sequence, herein designated as "RGS-GEF consensus 2," is
herein defined to be a consensus sequence of AA,-AAA-AA3-AA4-(gap of 13 amino
acids)-
AA,g-AAi9,wherein:
AA, is A;
AA2 is V or P;
AA; is L;
AA4 is either S or a gap of one amino acid, contiguous with the gap of 13
amino acids;
AA,B is either R or W;
AA,9 is either V or Y.
Other proteins, including other GEF proteins can be aligned with the RGS
domain of RGS
proteins as shown in Figure 18, and using methods described herein, to
determine if they
contain a sub-RGS consensus sequence, such as RGS-GEF consensus I or RGS-GEF
consensus 2, as defined above.
In examining Figure 18 it is also apparent that a nucleotide sequence uniques
to RGS
proteins that are not GEF proteins is shown by the nucleotide sequences which
encode the
amino acids that correspond to the 13-14 amino acid gap in RGS-GEF proteins.
These
nucleotide sequences could be used as probes to identify particular types of
RGS proteins.
RGS-GEF polypeptides are preferably biologically-active. By biologically-
active, it
is meant that a polypeptide fragment possesses an activity in a living system
or with
components) of a living system. Biological-activities include, but are not
limited to a
specific binding affinity for a G protein a subunit, as defined above, and GAP
activity
toward a G protein a subunit. As described in the examples, such polypeptides
can be
prepared routinely, e.g., by recombinant,means or by proteolytic cleavage of
isolated
polypeptides, and then assayed for a desired activity.
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A polypeptide of the present invention includes polypeptides which have less
than
100% identity to the amino acid sequences of pl 15 Rho-GEF (Fig. 10), Lsc
(Fig. 14),
KIAA0380 (Fig. 12), or DRhoGEF2 (Fig. 16). For the purposes of the following
discussion:
Sequence identity means that the same nucleotide or amino acid which is found
in the
sequences set forth in Fig. 10-17 is found at the corresponding position of
the compared
sequence(s). A polypeptide having less than 100% sequence identity to the
amino acid
sequences set forth in Figures 10, 12, 14, and 16 can be substituted in
various ways, e.g., by
a conservative amino acid. The sum of the identical and conservatively
substituted residues
divided by the total number of residues in the sequence is equal to the
percent sequence
similarity. For purposes of calculating sequence identity and similarity, the
compared
sequences can be aligned and calculated according to any desired method,
algorithm,
computer program, etc., including, e.g., FASTA, BLASTA. A polypeptide having
less than
100% amino acid sequence identity to the amino acid sequences of the GEF
proteins shown
in Figures 10, 12, 14, and 16 may comprise, for example, about 60, 65 percent
sequence
similarity and more preferably about 67, 70, 78, 80, 90, 92, 96, 99, etc.
percent sequence
amino acid sequence similarity.
In particular, the present invention relates to polypeptides, and
corresponding nucleic
acids, of p115, Lsc, KIAA380, and DrhoGEF2 which are mutated in the RGS domain
of a
GEF protein and which possess one or more of the RGS-GEF polypeptide
activities
mentioned above. By the term "mutated," it is meant herein that such sequences
are not
naturally-occurring. For example a mutated polypeptide as mentioned can have
one or more
naturally-occurring positions replaced by a conservative amino acid, e.g.,
(based on the size
of the side chain and degree of polarization) small nonpolar: cysteine,
proline, alanine,
threonine; small polar: serine, glycine, aspartate, asparagine; large polar:
glutamate,
glutamine, lysine, arginine; intermediate polarity: tyrosine, histidine,
tryptophan; large
nonpolar: phenylalanine, methionine, leucine, isoleucine, valine. Such
conservative
substitutions also include those described by Dayhoff in the Atlas of Protein
Sequence and
Structure 5 ( 1978), and by Argos in EMBO J., 8, 779-785 ( 1989). A
polypeptide having an
amino acid sequence as set forth in Figures 10, 12, 14, and 16 can be
substituted at 1, S, 10,
1 S, or 20 positions by conservative amino acids. The mutations can be
introduced into the
conserved consensus region or the other residues of the RGS domain of a GEF
protein.
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A mutation to an RGS-GEF polypeptide can be selected to have one or more of
the
activities mentioned above, e.g., a specific binding affinity for a G protein
a subunit, a GAP
activity toward a G protein a subunit, etc. Assays for such activities can be
conducted as
described below or as disclosed in Cerione and Zheng, 1996, Curr. Opin. In
Cell Biol.,
8:216.
An RGS-GEF polypeptide can be modified by introducing amino acid substitutions
into the hydrophobic core of the RGS domain (See Fig. 1, Panel A). For
example, a
conservative amino acid substitution would not be expected to affect activity,
whereas as
non-conservative amino acid substitution, e.g., changing a hydrophobic residue
to a
hydrophilic residue, would be expected to reduce ar eliminate its activity.
Hydrophobic
resiudes are nonpolar amino acids such phenylalanine, leucine, isoleucine,
valine, alanine,
methionine, tryptophan, and cysteine. Hydrophilic residues are polar amino
acids such as
lysine, arginine, histidine, glutamate, and aspartate.
Modifications to a RGS-GEF polypeptide of the present invention or
corresponding
nucleotide sequence, e.g., mutations, can also be prepared based on homology
searching
from gene data banks, e.g., Genbank, EMBL. Sequence homology searching can be
accomplished using various methods, including algorithms described in the
BLAST family
of computer programs, the Smith-Waterman algorithm, etc. For example,
conserved amino
acids can be identified between various sequences containing an RGS domain of
various
GEF proteins. (See Fig. 18} A mutations) can then be introduced into such
sequences by
identifying and aligning amino acids conserved between the polypeptides and
then
modifying an amino acid in a conserved or non-conserved position. A mutated
RGS-GEF
sequence can comprise conserved or non-conserved amino acids, e.g., between
corresponding regions of homologous nucleic acids. For example, a mutated
sequence can
comprise conserved or non-conserved residues from any number of homologous
sequences
as mentioned-above and/or determined from an appropriate searching algorithm.
Corresponding mutations can be made in specific regions of an RGS-GEF nucleic
acid. For example, mutations may be made wherein amino acids that particpate
in the
GTPase catalytic function or mutations may be made in amino acids that
function as contact
points between the RGS-GEF sequence and the Ga subunit.
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An RGS-GEF polypeptide or fragment thereof, or substituted RGS-GEF polypeptide
or fragment thereof, may also comprise various modifications, wherein such
modifications
include glycosylation, covalent modifications (e.g., of an R-group of an amino
acid), amino
acid substitution, amino acid deletion, or amino acid addition. Modifications
to the
polypeptide can'be accomplished according to various methods, including
recombinant,
synthetic, chemical, etc.
Polypeptides of the present invention (e.g., RGS-GEF polypeptides, and
fragments
and mutations thereof) may be used in various ways, e.g., as immunogens for
antibodies as
described below, as biologically-active agents (e.g., having one or more of
the activities
associated with an RGS-GEF polypeptide), as inhibitors of the activities of
the
corresponding full-length polypeptide. For example, upon binding of p 115 Rho-
GEF to the
Ga subunit, a cascade of events is initiated in the cell, e.g., promoting cell
proliferation
and/or cytoskeletal rearrangements. The interaction between p 115 Rho-GEF and
the Ga
subunit can be modulated by using a RGS-GEF polypeptide, or fragment thereof,
to inhibit
the interaction between p 115 Rho-GEF and the Ga subunit. Such a fragment can
be useful
for modulating pathological conditions associated with the Rho signaling
pathway. A useful
fragment may be identified routinely by testing the ability of overlapping
fragments of the
entire length of the RGS domain of a GEF protein to inhibit the binding of p
115 Rho-GEF
with the Ga subunit or to inhibit the GAP activity of the p115 Rho-GEF toward
the Ga
subunit. The measurement of these activities is described below and in the
examples.
Peptides can be chemically-modified, etc.
A RGS-GEF polypeptide of the present invention can comprise one or more
structural domains, functional domains, detectable domains, antigenic domains,
and/or other
polypeptides of interest, in an arrangement which does not occur in nature,
i.e., not
naturally-occurring. A polypeptide comprising such features is a chimeric or
fusion
polypeptide. Such a chimeric polypeptide can be prepared according to various
methods,
including, chemical, synthetic, quasi-synthetic, and/or recombinant methods. A
chimeric
nucleic acid coding for a chimeric polypeptide can contain the various domains
or desired
polypeptides in a continuous or interrupted open reading frame, e.g.,
containing introns,
splice sites, enhancers, etc. The chimeric nucleic acid can be produced
according to various
methods. See, e.g., U.S. Pat. No. 5,439,819. A domain or desired polypeptide
can possess
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any desired property, including, a biological function such as catalytic,
signaling, growth
promoting, cellular targeting, etc., a structural function such as
hydrophobic, hydrophilic,
membrane-spanning, etc., receptor-ligand functions, and/or detectable
functions, e.g.,
combined with enzyme, fluorescent polypeptide, green fluorescent protein GFP
(Chalfie et
al., 1994, Science, 263:802; Cheng et al., 1996, Nature Biotechnology, 14:606;
Levy et al.,
1996, Nature Biotechnology, 14:610, etc. In addition, a RGS-GEF nucleic acid,
or a
fragment thereof, may be used as selectable marker when introduced into a host
cell. For
example, a nucleic acid coding for an amino acid sequence according to the
present
invention can be fused in frame to a desired coding sequence and act as a tag
for
purification, selection, or marking purposes. The region of fusion encodes a
cleavage site.
A polypeptide according to the present invention can be produced in an
expression
system, e.g., in vivo, in vitro, cell-free, recombinant, cell fusion, etc.,
according to the
present invention. Modifications to the polypeptide imparted by such system
include,
glycosylation, amino acid substitution (e.g., by differing codon usage),
polypeptide
processing such as digestion, cleavage, endopeptidase or exopeptidase
activity, attachment
of chemical moieties, including lipids, phosphates, etc. For example, some
cell lines can
remove the terminal methionine from an expressed polypeptide.
A polypeptide according to the present invention can be recovered from natural
sources, transformed host cells (culture medium or cells) according to the
usual methods,
including, ammonium sulfate or ethanol precipitation, acid extraction, anion
or cation
exchange chromatography, phosphocellulose chromatography, hydrophobic
interaction
chromatography, hydroxyapatite chromatography and lectin chromatography. It
may be
useful to have low concentrations (approximately 0.1-5 mM) of calcium ion
present during
purification (Price, et al., J. Biol. Chem., 244:917 (1969)). High performance
liquid
chromatography (HPLC) can be employed for final purification steps.
A RGS-GEF nucleic acid of the present invention can comprise the complete
coding
sequence for an RGS-GEF polypeptide, or fragments thereof. A nucleic acid
according to
the present invention may also comprise a nucleotide sequence which is 100%
complementary, e.g., an anti-sense, to any RGS-GEF nucleotide sequence.
A nucleic acid according to the present invention can be obtained from a
variety of
different sources. It may be obtained from DNA or RNA, such as polyadenylated
mRNA,
13
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WO 99/47557 PCT/US99/0605t
e.g., isolated from tissues, cells, or whole organism. The nucleic acid may be
obtained
directly from DNA or RNA, or from a cDNA library. The nucleic acid can be
obtained from
a cell at a particular stage of development, having a desired genotype,
phenotype (e.g., an
oncogenically transformed cell or a cancerous cell), etc. The nucleic acid may
also be
chemically synthesized.
A nucleic acid according to the present invention may include only coding
sequence
for an RGS-GEF polypeptide; coding sequence for an RGS-GEF polypeptide and
additional
functional coding sequences including, for example, leader sequences,
secretory sequences,
tag sequences (e.g. targeting tags, enzymatic tags, fluorescent tags etc.). A
nucleic acid
according to the present invention may also include coding sequence for an RGS-
GEF
polypeptide and non-coding sequences, e.g., untranslated sequences at either a
5' or 3' end,
or dispersed in the coding sequence, e.g., introns.
A nucleic acid according to the present invention may also comprise an
expression
control sequence operably linked to a nucleic acid as described above. The
phrase
IS "expression control sequence" means a nucleic acid sequence which regulates
expression of
a polypeptide coded for by a nucleic acid to which it is operably linked.
Expression can be
regulated at the level of the mRNA or polypeptide. Thus, the expression
control sequence
includes mRNA-related elements and protein-related elements. Such elements
include
promoters, enhancers (viral or cellular), ribosome binding sequences,
transcriptional
terminators, etc. An expression control sequence is operably linked to a
nucleotide coding
sequence when the expression control sequence is positioned in such a manner
to effect or
achieve expression of the coding sequence. For example, when a promoter is
operably
linked 5' to a coding sequence, expression of the coding sequence is driven by
the promoter.
Expression control sequences can be heterologous or endogenous to the normal
gene.
A nucleic acid in accordance with the present invention may be selected on the
basis
of nucleic acid hybridization. The ability of two single-stranded nucleic acid
preparations to
hybridize together is a measure of their nucleotide sequence complementarity,
e.g., base-
pairing between nucleotides, such as A-T, G-C, etc. The invention thus also
relates to
nucleic acids which hybridize to a nucleic acids comprising a nucleotide
sequence as set
forth in Figures 11, 13, 15, and 17. The present invention includes both
strands of nucleic
acid, e.g., a sense strand and an anti-sense strand.
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WO 99/47557 PCT/US99/06051
According to the present invention, a nucleic acid or polypeptide can comprise
one
or more differences in the nucleotide or amino acid sequence set forth in
Figures 10-17.
Changes or modifications to the nucleotide and/or amino acid sequence can be
accomplished
by any method available, including directed or random mutagenesis.
A nucleic acid coding for an RGS-GEF polypeptide according to the invention
may
comprise nucleotides which occur in a naturally-occurring GEF gene e.g.,
naturally-
occurring polymorphisms, normal or mutant alleles (nucleotide or amino acid),
mutations
which are discovered in a natural population of mammals, such as humans,
monkeys, pigs,
mice, rats, or rabbits. By the term naturally-occurring, it is meant that the
nucleic acid is
obtained from a natural source, e.g., animal tissues and cells, body fluids,
tissue culture
cells, forensic samples. Naturally-occurring mutations include deletions,
substitutions, or
additions of nucleotide sequence. These genes can be detected and isolated by
nucleic acid
hybridization according to methods well known to one skilled in the art. It is
recognized
that, by analogy to other oncogenes, naturally-occurring variants of GEF
proteins will
include variants with deletions, substitutions, and additions in the RGS
domain of a GEF
protein, which produce pathological conditions in the host cell and organism.
A nucleotide sequence coding for an RGS-GEF polypeptide of the invention may
contain codons found in a naturally-occurring gene, transcript, or cDNA, for
example, or it
may contain degenerate codons coding for the same amino acid sequences.
In addition, a nucleic acid or polypeptide of the present invention may be
obtained
from any desired mammalian organism, but also non-mammalian organisms.
Homologs
from mammalian and non-mammalian organisms can be obtained according to
various
methods. For example, hybridization with an oligonucleotide (see below)
selective for an
RGS domain of a GEF, or a RGS-GEF, of the present invention can be employed to
select
such homologs, e.g., as described in Sambrook et al., Molecular Cloning, 1989,
Chapter 11.
Such homologs may have varying amounts of nucleotide and amino acid sequence
identity
and similarity to previously identified RGS domain or RGS-GEF nucleotide or
polypeptide
sequence. Non-mammalian organisms include, e.g., vertebrates, invertebrates,
zebra fish,
chicken, Drosophila, yeasts (such as Saccharomyces cerevisiae), C. elegans,
roundworms,
prokaryotes, plants, Arabidopsis, viruses, etc.
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WO 99/47557 PCTNS99/06051
A nucleic acid according to the present invention may comprise, for example,
DNA,
RNA, synthetic nucleic acid, peptide nucleic acid, modified nucleotides, or
mixtures thereof.
A DNA can be double- or single-stranded. Nucleotides comprising a nucleic acid
can be
joined via various known linkages such as, for example, ester, sulfamate,
sulfamide,
phosphorothioate, phosphoramidate, methylphosphonate, carbamate, etc.,
depending on the
desired purpose. Linkages may be modified for purposes such as, for example,
resistance to
nucleases such as RNase H and improved in vivo stability. See, e.g., U.S. Pat.
Nos.
5,378,825.
Various modifications can be made to the nucleic acids, such as attaching
detectable
markers (avidin, biotin, radioactive elements), moieties which improve
hybridization,
detection, or stability. The nucleic acids can also be attached to solid
supports, e.g.,
nitrocellulose, nylon, agarose, diazotized cellulose, latex solid
microspheres,
polyacrylamides, etc., according to a desired method. See, e.g., U.S. Pat.
Nos. 5,470,967,
5,476,925, 5,478,893.
Another aspect of the present invention relates to oligonucleotides and
nucleic acid
probes. Such oligonucleotides or nucleic acid probes can be used, e.g., to
detect, quantify,
or isolate an RGS-GEF nucleic acid in a test sample. Detection can be
desirable for a
variety of different purposes, including research, diagnostic, and forensic.
For diagnostic
purposes, it may be desirable to identify the presence or quantity of a
specific RGS-GEF
nucleic acid sequence in a sample obtained from tissues, cells, body fluids,
etc. In a
preferred method, the present invention relates to a method of detecting a
target RGS-GEF
nucleic acid in a test sample comprising contacting the test sample with an
oligonucleotide
under conditions effective to achieve hybridization between the target and
oligonucleotide;
and detecting hybridization. An oligonucleotide in accordance with the
invention can also
be used in synthetic nucleic acid amplification such as PCR, e.g., Saiki et
al., 1988, Science,
241:53; U.S. Pat. No. 4,683,20, or or differential display (See, e.g., Liang
et al., ~ucl. ,Acid.
~,es., 21:3269-3275, 1993; USP 5,599,672; W097/18454). Oligonucleotides can be
identified routinely, e.g., to the DH, PH, and RGS-GEF domains to
differentially display
and/or amplify gene products containing such sequences.
Both sense and antisense nucleotide sequences are intended as part of the
invention.
A unique nucleic acid according to the present invention may be determined
routinely. An
16
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WO 99/47557 PCT/US99/06051
RGS-GEF nucleic acid may be used as a hybridization probe to identify the
presence of
RGS-GEF nucleotide sequence in a sample comprising a mixture of nucleic acids,
e.g., on a
Northern blot. Hybridization can be performed under stringent conditions to
select nucleic
acids having at least 95% identity (i.e., complementarity) to the probe, but
less stringent
conditions can also be used. A unique RGS-GEF nucleotide sequence can also be
fused in
frame, at either its 5' or 3' end, to various nucleotide sequences, including,
for example,
coding sequences for enzymes or expression control sequences, etc.
Hybridization can be performed under different conditions, depending on the
desired
selectivity, e.g., as described in Sambrook et al., Molecular Cloning, 1989.
For example, to
specifically detect RGS-GEF sequences, an oligonucleotide can be hybridized to
a target
nucleic acid under conditions in which the oligonucleotide only hybridizes to
the GEF
sequence from which the RGS -GEF sequence was derived, e.g., where the
oligonucleotide
is 100% complementary to the target. Different conditions can be used if it is
desired to
select target nucleic acids which have less than 100% nucleotide
complementarity, at least
about, e.g., 99%, 97%, 95%, 90%, 70%, 67%. Since a mutation in GEF genes can
cause
diseases or pathological conditions, e.g., cancer, benign tumors, an
oligonucleotide
according to the present invention can be used diagnostically. For example, a
patient having
symptoms of a cancer or other condition associated with the Rho signaling
pathway (see
below) can be diagnosed with the disease by using an oligonucleotide according
to the
present invention, in polymerase chain reaction followed by DNA sequencing to
identify
whether the sequence is normal, in combination with other oncogene
oligonucleotides, etc.,
e.g., p53, Rb, p21, Dbl, MTS1, Wtl, Bcl-l, Bcl-2, MDM2, etc.
Oligonucleotides according to the present invention can be of any desired
size,
preferably 14-16 oligonucleotides in length, or more. Such oligonucleotides
can have non-
naturally-occurring nucleotides, e.g., inosine. In accordance with the present
invention, the
oligonucleotide can comprise a kit, where the kit includes a desired buffer
(e.g., phosphate,
tris, etc.), detection compositions, etc. The oligonucleotide can be labeled
or unlabeled,
with radioactive or non-radioactive labels as known in the art.
Anti-sense nucleic acid can also be prepared from a nucleic acid according to
the
present, preferably an anti-sense RGS-GEF nucleotide sequence corresponding to
an RGS-
GEF nucleotide sequence of Figures 11, 13, 15, and 17. Anti-sense RGS-GEF
nucleic acid
17
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WO 99/47557 PCT/US99/06051
can be used in various ways, such as to regulate or modulate expression of GEF
proteins
containing RGS domains or to detect expression of RGS-GEF proteins, including
by in situ
hybridization. For the purposes of regulating or modulating expression, an
anti-sense
oligonucleotide may be operably linked to an expression control sequence.
The RGS-GEF nucleic acids according to the present invention can be labelled
according to any desired method. The nucleic acid can be labeled using
radioactive tracers
such as 32P, 35S~ ~2sh 3H~ or ~4C, to mention only the most commonly used
tracers. The
radioactive labeling can be carned out according to any method such as, for
example,
terminal labeling at the 3' or 5' end using a radiolabeled nucleotide,
polynucleotide kinase
(with or without dephosphorylation with a phosphatase) or a ligase (depending
on the end to
be labeled). A non-radioactive labeling can also be used, combining a nucleic
acid of the
present invention with residues having immunological properties (antigens,
haptens), a
specific affinity for certain reagents (ligands), properties enabling
detectable enzyme
reactions to be completed (enzymes or coenzymes, enzyme substrates, or other
substances
involved in an enzymatic reaction), or characteristic physical properties,
such as
fluorescence or the emission or absorption of light at a desired wavelength,
etc.
An RGS-GEF nucleic acid according to the present invention, including oligo-
nucleotides, anti-sense nucleic acid, etc., can be used to detect expression
of RGS-GEF
nucleic acids in whole organs, tissues, cells, etc., by various techniques,
including Northern
blot, PCR, in situ hybridization, etc. Such nucleic acids can be particularly
useful to detect
disturbed expression, e.g., cell-specific and/or subcellular alterations of
RGS-GEF
expression. The levels of RGS-GEF proteins can be determined alone or in
combination
with other genes products (oncogenes such as p53, Rb, Wtl, etc.), transcripts,
etc.
A nucleic acid according to the present invention can be expressed in a
variety of
different systems, in vitro and in vivo, according to the desired purpose. For
example, a
nucleic acid can be inserted into an expression vector, introduced into a
desired host, and
cultured under conditions effective to achieve expression of a polypeptide
coded for the
nucleic acid. Effective conditions includes any culture conditions which are
suitable for
achieving production of the polypeptide by the host cell, including effective
temperatures,
pH, medics, additives to the media in which the host cell is cultured (e.g.,
additives which
amplify or induce expression such as butyrate, or methotrexate if the coding
nucleic acid is
18
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WO 99/47557 PCT/US99/06051
adjacent to a dhfr gene), cyclohexamide, cell densities, culture dishes, etc.
A nucleic acid
can be introduced into the cell by any effective method including, e.g.,
calcium phosphate
precipitation, electroporation, injection, DEAE-Dextran mediated transfection,
fusion with
liposomes, and viral transfection. A cell into which a nucleic acid of the
present invention
has been introduced is a transformed host cell. The nucleic acid can be
extrachromosomal
or integrated into a chromosomes) of the host cell. It can be stable or
transient. An
expression vector is selected for its compatibility with the host cell. Host
cells include,
mammalian cells, e.g., COS-7, CHO, HeLa, LTK, NIH 3T3, yeast, insect cells,
such as Sf9
(S. frugipeda) and Drosophila, bacteria, such as E. coli, Streptococcus sp.,
Bacillus sp.,
yeast, fungal cells, plants, embryonic stem cells (e.g., mammalian, such as
mouse or
human), cancer or tumor cells Sf9 expression can be accomplished in analogy to
Graziani et
al., Oncogene, 7:229-235, 1992. Expression control sequences are similarly
selected for
host compatibility and a desired purpose, e.g., high copy number, high
amounts, induction,
amplification, controlled expression. Other sequences which can be employed
include
enhancers such as from SV40, CMV, inducible promoters, cell-type specific
elements, or
sequences which allow selective or specific cell expression.
A labelled polypeptide can be used, e.g., in binding assays, such as to
identify
substances that bind or attach to p 115 Rho-GEF, to track the movement of p
115 Rho-GEF
in a cell, in an in vitro, in vivo, or in situ system, etc.
A nucleic acid or polypeptide of the present invention can also be
substantially
purified. By substantially purified, it is meant that nucleic acid or
polypeptide is separated
and is essentially free from other nucleic acids or polypeptides, i.e., the
nucleic acid or
polypeptide is the primary and active constituent.
Another aspect of the present invention relates to the regulation of
biological
pathways in which a RGS-GEF polypeptide is involved, particularly pathological
conditions,
e.g., cell proliferation (e.g., cancer), growth control, morphogenesis, stress
fiber formation,
and integrin-mediated interactions, such as embryonic development, tumor cell
growth and
metastasis, programmed cell death, hemostasis, leucocyte homing and
activation, bone
resorption, clot retraction, and the response of cells to mechanical stress.
See, e.g., Clark
and Brugge, Science, 268:233- 239, 1995; Bussey, Science, 272:225-226, 1996.
Thus, the
invention relates to all aspects of a method of modulating an activity of a
RGS-GEF
19
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WO 99/47557 PCT/US99/06051
polypeptide comprising, administering an effective amount of an RGS-GEF
polypeptide or a
biologically-active fragment thereof, an effective amount of a compound which
modulates
the activity of a RGS-GEF polypeptide, or an effective amount of a nucleic
acid which codes
for a RGS-GEF polypeptide or a biologically-active fragment thereof. The
activity of the
RGS-GEF which is modulated may include binding to a Ga subunit or GAP activity
toward
a Ga subunit. The activity can be modulated by increasing, reducing,
antagonizing, or
promoting expression or activity of the RGS-GEF.
The present invention also relates to antibodies which specifically recognize
a RGS-
GEF polypeptide. Antibodies, e.g., polyclonal, monoclonal, recombinant,
chimeric, can be
prepared according to any desired method. For example, for the production of
monoclonal
antibodies, an RGS-GEF polypeptide according to Figures 10, 12,14, or 16 can
be
administered to mice, goats, or rabbit subcutaneously and/or
intraperitoneally, with or
without adjuvant, in an amount effective to elicit an immune response. The
antibodies can
also be single chain or FAb. The antibodies can be IgG, subtypes, IgG2a, IgGI,
etc.
An antibody specific for RGS-GEF means that the antibody recognizes a defined
sequence of amino acids within or including the amino acid sequence of the RGS
domain of
a GEF polypeptide. Thus, a specific antibody will bind with higher affinity to
an amino acid
sequence, i.e., an epitope, found in the RGS domain of a GEF polypeptdie than
to a different
epitope(s), e.g., as detected and/or measured by an immunoblot assay. Thus, an
antibody
which is specific for an epitope within or including the RGS domain of p 115
Rho-GEF is
useful to detect the presence of the epitope in a sample, e.g., a sample of
tissue containing
pl IS Rho-GEF gene product, distinguishing it from samples in which the
epitope is absent.
Additionally, in accordance with the present invention, ligands which bind to
an
RGS domain of a GEF polypeptide can also be prepared, e.g., using synthetic
peptide
libraries or aptamers (e.g., Pitrung et al., U.S. Pat. No. 5,143,854; Geysen
et al., 1987, J.
Immunol. Methods, 102:259-274; Scott et al., 1990, Science, 249:386; Blackwell
et al.,
1990, Science, 250:1104; Tuerk et al., 1990, Science, 249: 505.
Antibodies and other ligands which bind the RGS domain of a GEF polypeptide,
and
specifically antibodies and other ligands which bind the RGS domain of p 115
Rho GEF, can
be used in various Ways. These include, but are not limited to, uses
therapeutic, diagnostic,
and commercial research tools, e.g, to quantitate the levels of p115 Rho-GEF
polypeptide in
CA 02319037 2000-07-25
WO 99/47557 PCTNS99/06051
animals, tissues, cells, etc., to identify the cellular localization and/or
distribution of p 115
Rho-GEF, to purify p 115 Rho-GEF or a polypeptide comprising a part of p 115
Rho-GEF, to
modulate the function of p115 Rho-GEF, etc. Antibodies can be used in Western
blots,
ELIZA, immunoprecipitation, RIA, etc. The present invention relates to such
assays,
compositions and kits for performing them, etc.
An antibody according to the present invention can be used to detect
polypeptides or
fragments containing an RGS domain of a GEF polypeptide in various samples,
including
tissue, cells, body fluid, blood, urine, cerebrospinal fluid. A method of the
present invention
comprises contacting a ligand which binds to an RGS-GEF polypeptide of Figure
10, 12, 14,
or 16 under conditions effective, as known in the art, to achieve binding,
detecting specific
binding between the ligand and peptide. By specific binding, it is meant that
the ligand
attaches to a defined sequence of amino acids, e.g., within or including the
amino acid
sequence of the RGS domain as shown in Figures 10, 12, 14, and 16, or
derivatives thereof.
The antibodies or derivatives thereof can also be used to inhibit expression
of GEF proteins
containing an RGS domain. The levels of a GEF polypeptide containing an RGS
domain
may be determined alone or in combination with other gene products. In
particular, the
amount (e.g., its expression level) of the GEF polypeptide containing an RGS
domain can be
compared (e.g., as a ratio) to the amounts of other polypeptides in the same
or different
sample, e.g., p21, p53, Rb, WT1, etc.
A Iigand for the RGS domain of GEF polypeptides can be used in combination
with
other antibodies, e.g., antibodies that recognize oncological markers of
cancer, including,
Rb, p53, c-erbB-2, oncogene products, etc. In general, reagents which are
specific for the
RGS domain of GEF polypeptides can be used in diagnostic and/or forensic
studies
according to any desired method, e.g., as U.S. Pat. Nos. 5,397,712; 5,434,050;
5,429,947.
The present invention also relates to a transgenic animal, e.g., a non-human-
mammal, such as a mouse, comprising an RGS-GEF polypeptide. Transgenic animals
can
be prepared according to known methods, including, e.g., by pronuclear
injection of
recombinant genes into pronuclei of 1-cell embryos, incorporating an
artificial yeast
chromosome into embryonic stem cells, gene targeting methods, embryonic stem
cell
methodology. See, e.g., U.S. Patent Nos. 4,736,866; 4,873,191; 4,873,316;
5,082,779;
5,304,489; 5,174,986; 5,175,384; S,I75,385; 5,221,778; Gordon et al., Proc.
Natl. Acad.
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WO 99/47557 PCTNS99/06051
Sci., 77:7380-7384 (1980); Palmiter et al., Cell, 41:343-345 (1985); Palmiter
et al., Ann.
Rev. Genet., 20:465-499 (1986); Askew et al., Mol. Cell. Bio., 13:4115-4124,
1993; Games
et al. Nature, 373:523-527, 1995; Valancius and Smithies, Mol. Cell. Bio.,
11:1402-1408,
1991; Stacey et al., Mol. Cell. Bio., 14:1009-1016, 1994; Hasty et al.,
Nature, 350:243-246,
1995; Rubinstein et aL, Nucl. Acid Res., 21:2613-2617,1993. A nucleic acid
according to
the present invention can be introduced into any non-human mammal, including a
mouse
(Hogan et al., 1986, in Manipulating the Mouse Embryo: ~ A Laboratory Manual,
Cold
Spring Harbor Laboratory, Cold Spring Harbor, New York), pig (Hammer et al.,
Nature,
315:343-345, 1985), sheep (Hammer et al., Nature, 315:343-345, 1985), cattle,
rat, or
primate. See also, e.g., Church, 1987, Trends in Biotech. 5:13-19; Clark et
al., 1987, Trends
in Biotech. 5:20-24; and DePamphilis et al., 1988, BioTechniques, 6:662-680.
Additionally,
custom transgenic rat and mouse production is commercially available. These
transgenic
animals are useful, for example, as a cancer model or as a model to evaluate
the effects of
overexpression of the RGS-GEF polypeptide.
Generally, the nucleic acids, polypeptides, antibodies, etc. of the present
invention
can be prepared and used as described in, U.S. Pat. Nos. 5,501,969, 5,506,133,
5,441,870;
WO 90/00607; WO 91/15582;
Other aspects of this invention relate to methods to assay for, or identify,
molecules
that modulate the following interactions and effects: the interaction between
an RGS domain
of a GEFand its cognate binding substrate; the interaction between an RGS
domain of a
GEF and a Ga subunit; the effect of G protein subunit stimulation on a guanine
nucleotide
exchange activity of a GEF protein containing an RGS domain; the effect of a
GEF protein
having an RGS domain as a GTPase activating protein for a G protein subunit.
Activity can be modulated in various ways, e.g., enhancing, activating,
stimulating,
suppressing, preventing, inhibiting, etc. A modulatory molecule can be an
agonist,
antagonist, or have partial activities thereof. Modulating molecules can be
any type of
molecule, including but not limited to small molecules, proteins, peptides,
antibodies,
nucleic acids, etc. 'In general, a compound having an in vitro activity will
be useful in vivo
to modulate a biological pathway associated with a GEF protein containing an
RGS domain,
e.g., to treat a pathological condition associated with the biological and
cellular activities
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WO 99/47557 PCT/US99/06051
mentioned above. The modulatory molecules can comprise a mixture of the same
or
different molecules.
A binding substrate for the RGS domain of a GEF protein can be any material to
which the RGS domain binds specifically, including members of the Gal2 family.
See,
e.g., Strathman and Simon, Proc. Natl. Acad Sci., 88:5582, 1991. For example,
a method
of identifying or assaying for a molecule that modulates or regulates the
binding of a G
protein a subunit to a GEF protein containing an RGS domain, such as pl 15 Rho-
GEF, can
be conducted in accordance with this invention. In one embodiment, a GTP bound
a
subunit, or derivative thereof, is incubated with a GEF protein, or fragment
thereof,
containing the RGS domain, in the presence and absence of a test molecule to
determine
whether the presence of the test compound modulates the binding between the
GEF protein
and the G protein a subunit. The incubation is accomplished under effective
conditions,
i.e., conditions under which binding or attachment occurs. Binding can be
detected in one
or more ways. For example, the GEF protein or the binding substrate is labeled
detectably;
the labelled bound component is separated from the labelled free component;
and the
amount of bound-detectably labeled GEF protein or binding substrate
determined. The
detectable label can be of any desired composition, e.g., radioactive,
fluorescent, etc. Such
an assay can be performed in either solid or liquid phase.
In one aspect of the invention, it is desirable to identify molecules that
regulate the
binding of the Gale family of subunits, eg. Ga,2 and Ga~3, with a GEF, e.g.,
pl 15 Rho GEF,
Lsc, KIAA380, or DrhoGEF2. The assay can be conducted using a complete GEF
protein,
or any subfragments thereof which contain the RGS domain, or biologically
active
subfragments of the RGS domain. The assay is typically conducted with stable
analogs of
the GTP bound state of the Ga subunit, including a subunits bound to either
GDP-AIF4 or
GTFyS. For example, a binding assay may be conducted by the procedure
described in
Example 5 below wherein a COS cell is transfected with a nucleic acid
construct for a myc-
tagged polypeptide, such as p 115 Rho GEF, or a fragment thereof and complexes
of the
polypetide and a Ga subunit are detected by precipitation of any bound complex
with a first
antibody to one of components and detection of the amount of a second bound
component
with a second antibody. Binding assays could also be performed using
techniques that are
23
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WO 99/47557 ~ PCTNS99/06051
well known in the art such as by binding one of the components to a column and
then
determining the amount of a second labelled compoent that binds to the column.
Relevant
assay methods are also disclosed, for example, in Berman et al, 1996, J. Biol.
Chem.
271:27209.
A method of isolating or assaying for a molecule that modules or regulates the
stimulatory effect of a RGS-GEF polypeptide on GTPase activity, such as a
GTPase activity
of a Ga subunit, can also be conducted in accordance with the invention. For
example, a
Ga subunit is incubated under effective conditions with an RGS-GEF polypeptide
having
GTPase stimulatory effect in the presence and absence of a test inhibitor to
determine
whether the presence of the test inhibitor modulates its stimulatory effect.
The assay can
conducted using a complete RGS-GEF polypeptide, a GEF protein, or any
subfragments
thereof which contain the RGS domain, or biologically active subfragments of
the RGS
domain. An RGS-GEF polypeptide can be p 115 Rho GEF, Lsc, KIAA380, DrhoGEF2,
or
biologically-active fragments thereof. For example, an assay can be conducted
using a p 115
Rho GEF in conjuction with an alt or an a13 subunit, as described in the
examples
discussed herein, as well as using other variations or assay methods which are
well known in
the art. For example, the assay may be conducted in accordance with Example 5
below, in
which Ga subunits were loaded with ['y 32P]GTP and the amount of hydrolysis
under various
conditions, including the presence of an RGS-GEF polypeptide, was determined
by
measuring the amount of 32Pi in the supernatant after centrifugation of the
assay mixture.
Relevant assay methods are also disclosed, for example, in Berman et al, 1996,
J. Biol.
Chem. 271:27209.
A method of identifying or assaying for a moiecule that modulates the
stimulatory
effect of an activated Ga subunit on a RGS-GEF polypeptide having GEF mediated
nucleotide exchange for a monomeric G protein can also be conducted in
accordance with
this invention. For instance, a first assay can be conducted by incubating an
activated Ga
alpha subunit with a GEF protein (e.g., pl 15 Rho GEF, Lsc, KIAA380, DrhoGEF2,
or
biologically-active fragments thereof, which retain GEF activity) and a
monomeric G
protein in the presence and absence of a test modulator to determine whether
the test
modulator has an inhibitory, enhancing, etc. effect on the ability of an
activated Ga subunit
24
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WO 99/47557 PCT/US99/06051
to stimulate GEF mediated nucleotide exchange of a monomeric protein. See e.g.
Hart et al.,
1996, J. Biol. Chem. 221:25452. The test modulator can be further evaluated by
conducting
a second assay in which said GEF protein and a monomeric G protein, without
the G protein
subunit, are incubated in the presence or absence of the test modulator to
determine whether
the test modulator had any effect on GEF mediated nucleotide exchange of the
monomeric
protein, and then comparing the modulation effect in the first and second
assays to
determine whether the modulating effect in the first assay is different from
the modulating
effect in the second assay, thereby indicating that the test modulator
modulates the
interaction of an activated Ga subunit with the GEF protein rather than the
interaction of the
GEF protein with the monomeric G protein. For example, the stimulatory effect
on GEF
mediated guanine nucleotide exchange may be measured according to Example 6
below,
wherein RhoA was loaded with [3H]GDP and the dissociation of GDP from RhoA was
measured under various conditions by the determination of bound GDP by
filtration, prior to
an after incubation. (See e.g. Northrop et al., J. Biol. Chem., 257, 11416-
11423 (1982)).
1 S A method of identifying a molecule that mimics the stimulatory effect of
an activated
Ga subunit on GEF mediated nucleotide exchange of a monomeric G protein may
also be
conducted in accordance with the invention. The method comprises identifying a
test
compound that exhibits a binding affinity for the RGS domain of GEF proteins
and then
incubating a GEF protein and monomeric G protein in the presence or absence of
the test
compound to determine whether the test compounds exhibits a stimulatory effect
on GEF
mediated nucleotide exchange of a monomeric G protein. The identification of
test
compounds that exhibit a binding affinity for the RGS domain of GEF proteins
may be
accomplished using techniques well knoEVn in the art. For example, an RGS
polypeptide
may be bound to a column and cocktails of test compounds may be passed over
the column
to determine if any were selectively bound by the column.
A method of identifying a molecule, or mixture of molecules, that mimics the
stimulatory effect of an RGS domain of GEF polypeptide on Ga subunit GTPase
activity
may also be conducted in accordance with the invention. The method comprises
identifying
a test compound that exhibits a binding affinity for a Ga subunit and
incubating a GTP
loaded Ga subunit in the presence or absence of the test compound to determine
whether the
test compound exhibits a stimulatory effect GTPase activity of the Ga subunit.
The
CA 02319037 2000-07-25
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identification of test compounds that exhibit a binding affinity for the G a
subunit may be
accomplished using techniques well known in the art. For example, a Ga,2 may
be bound
to a substrate and incubated with both a GEF polypeptide containing an RGS
domain and
the test compound to determine whether the test compound competes with the RGS
domain
for binding to the Ga subunit.
The modulation of oncogenic transforming activity by an RGS-GEF component, or
derivatives thereof, can be measured according to various known procedures,
e.g., Eva and
Aaronson, Nature, 316:273-275, 1985; Hart et al., J. Biol. Chem., 269:62-65,
1994. A
compound can be added at any time during the method (e.g., pretreatment of
cells; after
addition of the RGS-GEF, etc.) to determine its effect on the oncogenic
transforming
activity of the RGS-G~EF component. Various cell lines can also be used.
Other assays for monomeric GTPase-mediated signal transduction can be
accomplished according to the invention by analogy to procedures known in the
art, e.g., as
described in U.S. Pat. Nos. 5,141,851; 5,420,334; 5,436,128; and 5,482,954;
W094/16069;
IS W093/16179; W091/15582; W090/00607.
The present invention thus also relates to the treatment and prevention of
diseases
and pathological conditions associated with signal transduction mediated by
GEF proteins
that contain an RGS domain, e.g., cancer, diseases associated with abnormal
cell
proliferation. For example, the invention relates to a method of treating
cancer comprising
administering, to a subject in need of treatment, an amount of a compound
effective to treat
the disease, where the compound is a regulator of the stimulatory effect of
GEF protein
containing an RGS on Ga subunit GTPase activity or where the compound is a
regulator of
the stimulatory effect of a Ga subnit on GEF mediated nucleotide exchange by a
monomeric
GTPase. Treating the disease can mean, delaying its onset, delaying the
progression of the
disease, improving or delaying clinical and pathological signs of disease. A
regulator
compound, or mixture of compounds, can be synthetic, naturally-occurnng, or a
combination. A regulator compound can comprise amino acids, nucleotides,
hydrocarbons,
lipids, polysaccharides, etc. A regulator compound is preferably a compound
that regulates
expression of a GEF protein containing an RGS domain, e.g., inhibiting or
increasing its
mRNA, protein expression, or processing, or a compound that regulates the
interaction of
the RGS domain of the GEF protein with a Ga subunit. To treat the disease, the
compound,
26
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WO 99/47557 PCT/US99/06051
or mixture, can be formulated into pharmaceutical composition comprising a
pharmaceutically acceptable carrier and other excipients as apparent to the
skilled worker.
See, e.g., Remington's Pharmaceutical Sciences, Eighteenth Edition, Mack
Publishing
Company, 1990. Such composition can additionally contain effective amounts of
other
compounds, especially for treatment of cancer.
EXAMPLES
Example 1. Identification of homoloQV between a Rho GEF and~roteins which
regulate G
protein si haling.
The RGS family of proteins act as negative regulators of G protein signalling.
Nineteen mammalian members of the family have been identified, all of which
encode
proteins that contain a homologous core domain called the RGS box.
Examination of the sequence of p 115-GEF, a GEF specific for Rho, revealed an
N-
terminal region with specific homology to the conserved domain of RGS
proteins, including
RGS4, RGS2, GAIP, RGS 12, and RGS 14 (Fig. 1 ). Analysis of three other Rho
GEF
proteins, Lsc, KIAA380, and DrhoGEF also showed that they contained regions of
specific
homology to the conserved domain of RGS proteins (Fig. 1 ).
The crystal structure of a complex between RGS4 and A1F4-activated Goc;~
revealed
that the functional core of RGS4 (the RGS box) contains nine a-helixes that
fold into two
small subdomains (Tesmer et al., Cell, 89, 251 (1997)). The RGS box has been
shown to
contain the GAP activity towards Ga subunits (Popov et al., Proc. Natl. Acad.
Sci. USA, 94,
7216 ( 1997)). The hydrophobic core residues of the box, which are conserved
in members
of the RGS family, are important for stability of structure and GAP activity
(Tesmer et al.,
Cell, 89, 251 (1997) and Srinivasan et al., J. Biol. Chem., 273, 1529 (199$).
RGS4
stimulates the GTPase activity of Ga;, by interacting with its three switch
regions, primarily
by stabilization of the transition state of GTP hydrolysis (Tesmer et al.,
Cell, 89, 251
( 1997)).
Most of the hydrophobic residues that form the core of the RGS domain are
conserved in p 115 Rho GEF ( 17 out of 23) (Fig. 1 ). The position of gaps in
the alignment
correspond to the loops between alpha helixes of RGS domain structure. This
homology
suggested that the N-terminal region of p 115-GEF may have a similar structure
to the RGS4
box domain and possess GAP activity. In contrast, the residues of RGS4 that
make contact
27
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WO 99/47557 PCT/US99/06051
with the switch regions of Ga;,(GDP-A1F4-) are not well conserved, and any GAP
activity of
p115 Rho GEF will have a unique mechanism or a significantly different
specificity than
those previously identified.
A search of the gene bank revealed three other Rho-GEF members that have
regions
homologous to the RGS region of pl 15. These include Lsc, KIAA380, and
DrhoGEF2 (Fig.
1 ). Lsc appears to be the mouse homolgue of p 115 Rh0 GEF and KIAA380 appears
to be
the human homolgue of Drosophila DrhoGEF2 (Whitehead et al., J. Biol. Chem.,
271,
18643 ( 1996); Barrett et al., Cell, 91, 905 ( 1997)}. These four Rho-GEF's
define a new
RGS related family of proteins which also possess guanine nucleotide exchange
activtity for
Rho.
An alignment of RGS domains of the four GEF proteins known to contain RGS
domains (p115 Rho GEF, Lsc, KIAA380, DRhoGEF2) with the RGS domains of RGS
proteins RET-RGS 1, RGS 1, RGS2, RGS3, RGS4, RGS7, RGS 10, RGS 12, RGS 14,
Rapl/2B.P., and GAIP shows that a novel sub-RGS consensus sequence is defined
by the
RGS sequence of the four GEF proteins (Fig. 18). As shown in the bottom set of
sequences
shown in Fig. 18, a novel sub-RGS consensus sequence is shown by the large gap
of 13 to
14 amino acids in the homology alignment, along with the conservation of amino
acids on
either side of the gap.
Example 2. The RHO GEF protein pl 15 RHO-GEF stimulates the GTFase activit
~~of
Ga~3 and Ga,2 subunits.
P115 Rho GEF was tested to determine it's capability in stimulating the
intrinsic
GTPas;, activity (GAP activity) of Ga,3 and Ga,2.
Ga,2 was expressed in Sf9 cells and purified as described in Kozasa and
Gilman, J.
Biol. Chem., 270, 1734 (1995). Ga,~ was prepared by a similar procedure using
the
previously described baculovirus method (Singer and Miller, J. Biol. Chem.,
269, 19796
( I 994)) and octylglucoside during washing and elution of the a subunit after
immobilization
of the heterotrimer on Ni-NTA resin (Qiagen}. The eluted Ga,3 was further
purified by
absorption to and elution from hydroxyapatite. Ga,2 or Gala (20-30 pmol) was
loaded at
30°C for 30 or 40 minutes, respectively with 5 N,M['y 32P]GTP (50-100
cpm/fmol) and in the
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WO 99/47557 PCTNS99/06051
presence of 5 mM EDTA. Samples were then rapidly filtered by centrifugation at
4°C
through Sephadex G50 which had been equilibrated with buffer A (SO mM NaHepes
(pH
8.0), 1 mM dithiolthreitol, 5 mM EDTA, and 0.05% polyoxyethylene 10-
laurylether) to
remove free [y 32P]GTP and [32Pi]. Hydrolysis of GTP was initiated by adding
Ga loaded
with ['y 32P]GTP in buffer A containing 8 mM MgS04, 1 mM GTP and the indicated
amount
of pI 15. The reaction mixture was incubated at 4°C or 15°C.
Aliquots (50 p,I) were
removed at the indicated times and mixed with 750 ~1 of 5%(w/v) NoritA in 50
mM
NaH2P04. The mixture was centrifuged at 2000 rpm for 5 minutes and 400 p,l of
supernatant containing 32Pi were counted by liquid scintillation spectrometry.
The hydrolysis of GTP bound to Ga,3 and Ga,~ was performed at 15°C
either with
or without IOnM full-length p115 (Fig. 2, Panel A). The hydrolysis of GTP
bound Ga,3 and
Gait was measured at 4°C in the presence of various concentrations of
p115 (Fig. 2, Panel
B). Full-length p115 was able to stimulate a single round of hydrolysis of [Y
32P]GTP which
had been prebound to the Ga,3 subunit. The intrinsic GTPase activity of Ga~2,
the closest
homologue of Ga,3, was also stimulated by full-length p115. At 15°C,
the k~~ for
hydrolysis of GTP by Ga, 2 (0.07 min'' ) and Ga, 3 (0.24 min' ~ } were
respectively increased
5-fold and 10-fold by 10 nM p115 (Fig. 2, Panel A). Similar results were
obtained with
several preparations of Ga,2 and Ga,3. Treatment of pl 15 at 90°C
inactivated this GAP
activity. Due to the rapid hydrolytic rates of Ga,3, assays were performed at
4°C to better
estimate the effect of p 115 on the initial rate of GTPase activity by the G
protein (Fig. 2,
Panel B). Under these conditions, 100 nM pl 15 caused and 80-fold increase in
the GTPase
activity of Ga,3. In contrast, the hydrolytic rate of Ga,2 was increased only
6-fold.
Although stimulation of both proteins was observed at concentrations of p I 15
as low as 1
nM, measurements at both temperatures indicate that p115 is a more efficacious
GAP for
Ga,3 than Ga,2.
In the absence of a receptor, the rate limiting step in the binding of GTPyS
to Ga and
the steady state hydrolysis of GTP is the release of GDP. P 115 did not affect
either the rate
of GTP~yS binding to Gait and Ga,3 or the steady state of GTPase activity of
either subunit.
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WO 99/47557 PCT/US99/06051
Therefore, p 115 stimulates only the intrinsic GTPase activity of Ga, ~ and
Ga; 3 without
effecting their rates of nucleotide exchange.
The conserved RGS box region of RGS proteins is sufficient to show GAP
activity
in vitro (Popov et al., Proc. Natl. Acad. Sci. USA, 94, 7216 ( 1997)):
Therefore, a fusion
protein (Fig. 1, Panel B) of glutathione-S-transferase and the N-terminal
region of p115,
GST-RGS, was tested for GAP activity. This region retains RGS homology domain
but not
the Dbl or PH domains of p 115. This "RGS domain" of ~p 115 ( 10 nM) was
almost as active
as full-length p115 when tested for GAP activity for Ga;2 and Ga,3 (Fig. 3).
In contrast, a
construct of p 115 missing this N-terminal region was ineffective. Thus, the
data indicates
that the RGS homology region is responsible for the GAP activity of p 115.
Example 3. The n 115 RHO-GEF, does not stimulate the GTPase activity of Ga;
GaZ Gay
and Gas subunits.
The specificity of the GAP activity of p 115 for various G protein a subunits
was
examined as follows.
Gas was expressed in and purified from Escherichia coli as described in Lee et
al.,
Meth. Enrymol., 237, 146 (1994). Ga;, GaZ, and GocqR183C were expressed in Sf9
cells and
purified as described in Kozasa and Gilman, J. Biol. Chem., 270, 1734 (1995)
and
Biddlecome et al., J. Biol. Chem., 271, 7999 ( 1996). Ga;, Gaz, and GaZ were
loaded with 5-
10 ~t.M ('y 32P]GTP at 20°C (for Gas) or 30°C (for Ga; and GaZ)
for 20 minutes in the
presence of 5 rriM EDTA and GAP assays were performed as described above for
Ga;2 and
Ga;3. Gap activity on Gaq was assessed with a mutant Ga~R183C. An analogous
mutation
in Ga; R1178C, causes markedly reduced GTPase activity but response to RGS
proteins was
retained (Berman et al., Cell, 86, 445 ( 1996)). The slow GTPase activity of
GagR 183C
enables loading of [y 32P)GTP on Gotq without using receptor to accelerate
nucleotide
exchange. GotqR 183C was loaded with 10 N,M ['y 32P)GTP in the presence of 50
mM Hepes
(pH7.4), 0.1 mg/ml BSA, 1 mM DTT, 1 mM EDTA, 0.9 mM MgS04, 30 mM (NH4)2S04,
4% glycerol, and 5.5 mM CHAPS at 20°C for 2 hours. The reaction mixture
was rapidly
CA 02319037 2000-07-25
WO 99/4?55? PCT/US99/06051
filtered through Sephadex G50 which had been equilibrated with 50 mM Hepes (pH
7.4), I
mM DTT, 1 mM EDTA, 0.9 mM 504, 0.1 mg/ml BSA, and I mM CHAPS.
The results of this study showed that pl 15 (100 nM) did not stimulate the
GTPase
activity of Ga;, GaZ, or Gay under conditions where RGS4 acts as a GAP for
these Ga
subunits (Figure 4). Similarly, p115 did not accelerate the GTPase activity of
Gas, nor did
p I 15 Rho GEF have any GAP activity towards RhoA or rac 1. Thus, p 115 is a
GAP with
specificity for Ga,2 and Ga,3.
Example 4. Selective inhibition of p115 GAP activit~y AIF4 activated forms of
Ga
subunits.
RGS proteins have been shown to have high affinity for the GDP-A1F~ bound form
of a subunits, a configuration similar to the transition state of GTP
hydrolysis (Tesmer et al.,
Cell, 89, 251 ( 1997), Berman et al., J. Biol. Chem., 271, 27209 ( 1996)).
Therefore, the
GDP-AIF4 forms of Ga should compete with GaGTP for interaction with p 1 I5 and
block
the observed GAP activity. As shown in Fig. 5, Panel A, GDP-AIFa bound Gait
and Ga,3
effectively inhibited the GAP activity of p115 for Gait, while similar forms
of Gas, Ga;,
and Gocq were without effect. Additionally, a tritration of GDP-AIF,~ bound
forms of Ga,2
and Ga" demonstrated that the subunits are equipotent in inhibiting the GAP
activity of
Ga,3 (Fig. 5, Panel B). These competition assays suggest that the two G
protein subunits
have a similar affinity for pl 15 and supports the apparent differential
efficacy of p115
towards the subunits as shown in Fig. 2.
Example 5. Bindin og f Ga,3 to pl 15 Rho GEF in vivo.
The following experiments demonstrated that Ga, 3 and p 1 I 5 Rho GEF interact
in a
GTP-dependent manner.
EXV-myc tagged (for COS cell transfections) and pAc-Glu tagged (for
baculovirus
expression) proteins with deletions of the RGS or DH domains were constructed
as
previously described in Hart et al., J. Biol. Chem., 271, 25452-25458 (1996).
Full-length
versions were constructed in the same vectors. A fusion of GST to the first
246 amino acids
of p 115 Rho GEF was constructed in pGEX4T-2 (Pharmacia). Transfections,
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WO 99/47557 PCT/US99/06051
immunoprecipitations, and puriflcations were performed as previously described
in Hart et
al., J. Biol. Chem., 271, 25452-25458 (1996).
In COS cells transfected with myc-tagged p115 Rho GEF, Ga,3 can be
specifically
immunoprecipitated using the anti-myc antibody (Fig. 6, Panels A and B). This
interaction
is dependent on the presence of aluminum fluoride which is added to mimic the
activated
GTP-bound state of the Ga,3. Additionally, a truncated mutant of p115 Rho GEF
which
lacks the amino-terminal RGS domain is incapable of mediating co-
immunoprecipitation,
while full-length protein with a deletion in the DH domain does mediate co-
imlnunoprecipitation. The differential binding of full-length and truncated
Rho GEF
proteins could also be detected using antibodies to Ga, 3 to immunoprecipitate
the complex
(Fig. 6, Panel C). A very weak interaction with Ga,2 was detectable, while
antibodies to
Gas, Ga;, Gocq and GaZ do not detect immunoreactive bands in the anti-myc
immunoprecipitates, in spite of the fact that their respective antigens are
detectable in the
whole cell lysates. The co-immunoprecipitation of p115 Rho GEF and Gai3 can be
reproduced in a semi-purified system in which purified Ga,3 is added to
immunoprecipitated
p 115 Rho GEF (Fig. 6, Panel D), suggesting a direct interaction. This direct
interaction is
consistent with the observation that p115 Rho GEF stimulates Ga,3 GTPase
activity, but
also indicates that pl 15 Rho rnay be an effector of Ga~3.
Binding could also be detected between the Rho GEF protein, KIAA380 and the
ai2
G protein subunit (Fig. 9, KIAA380 is referred to as FL147). In COS cells
transfected with
myc-tagged KIAA380, Ga,2 can be specifically immunoprecipitated using the anti-
myc
antibody (Fig. 9.. Panels A and B, KIAA380 is referred to as FL147). Tbis
interaction is
dependent on the presence of aluminum fluoride which is added to mimic the
activated
GTP-bound state of the Ga,3.
Example 6. Stimulation of p115 Rho GEF activity by Gai3.
The ability of Ga, 3 to affect the exchange activity of p 115 Rho GEF was
examined
by incubating RhoA and p115 Rho GEF with or without Ga,3 to determine the
effect on
guanine nucleotide exchange.
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RhoA (2.5 p.M) was loaded with [3H)GDP by incubation at 30°C for 1 hour
with 25
~tM GDP ( 10,000 cpm/pmol) in 50 mM NaHepes, pH 7.5, 50 mM NaCI, 4 mM EDTA,
1 mM dithiolthreitol and 0.1 %Triton X-100. After addition of MgCl2 to 9 mM
and
octylglucoside to 1 %, the Rho was incubated for an additional 5 minutes and
separated from
free GDP by rapid filtration through Sephadex-G50 that had been equilibrated
with 50 mM
NaHEPES, pH 7.5, 50 mM NaCI, 1 mM EDTA, 1 mM dithiolthreitol, 5 mM MgCl2, and
1 %
octylglucoside. Dissociation of GDP from RhoA was measured at 30°C in
20 p,l of 50 mM
NaHEPES, pH 7.5, 50 mM NaCI, 1 mM EDTA, 1 mM dithiolthreitol, 5 mM MgCl2, 30
mM
A1C13, S mM NaF, and 5 p,M GTPyS. Unless specified, G protein alpha subunits
were
preincubated with AMF (30 p,M A1C1~, 5 mM MgCl2 and 5 mM NaF) prior to mixing
with
other proteins. Where indicated, alpha subunits were treated with 25 p,M GTPyS
or GDP~3S
rather than AMF and reactions were incubated without AMF but with 5 ~tM of the
respective nucleotide. Reactions were started with the addition of [3H]-GDP-
RhoA and
bound GDP was determined by filtration (Northup et al., J. Biol. Chem., 257,
11416-11423
I S ( 1982)) prior to and after incubation.
The Gas and Gtx; alpha subunits were purified after expression in Escherichia
coli
(Lee et al., Meth. Enzymol., 237, 146-164 (1994)). The Gaq and GaZ alpha
subunits were
coexpressed in Sf9 cells with hexahistidine-tagged beta and gamma subunits and
isolated as
described (Kozasa and Gilman, J. Biol. Chem., 270, 1734-1741 ( 1995)). Gay ~
was prepared
by a similar procedure to Gait using baculovirus (Singer et aL, J. Biol.
Chem., 269, 19796-
19802 ( 1994)) and octylglucoside during washing and elution of the a subunit
after
immobilization of the heterotrimer on Ni-NTA resin (Qiagen). The eluted Ga,3
was further
purified by absorption to and elution from hydroxyapatite. About 500 ug of
purified Ga,3
can be obtained from 3 liters of cells. The expression of GST-RhoA in SP9
cells, cleavage
of the GST tag and isolation of the free RhoA were as described in Singer et
al., J. Biol.
Chem., 271, 4505-4510, ( 1996).
These studies demonstrated that the Ga» is capable of stimulating the activity
of
full-length p115 Rho GEF in a manner which depends on the concentrations of
both pl 15
Rho GEF (Fig. 7, Panel A) and Ga,3 (Fig. 7, Panel B). The closely related
alpha subunit
33
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WO 99/47557 PCTNS99/06051
Ga,2 was ineffective in stimulating the activity of pl 15 Rho GEF in these
experiments (Fig.
7, Panel A). Stimulation of Rho exchange was also monitored as a function of
the activation
state of Ga,3. The data graphed in Fig. 7, Panel C confirm that the
stimulation of exchange
activity is dependent on either aluminum fluoride (AMF) or GTPyS, but is not
stimulated by
the deactivated nucleotide state mimicked by GDP~S. Additionally, a series of
other alpha
subunits including Gocq, GaZ, Gas, and Goc; also did not effect the activity
of p 115 Rho GEF
(Fig. 7, Panel D). These results are consistent with with the activated Ga,3 -
dependent
binding shown in Figure 6, and suggest that the productive binding of Ga,~ to
p115 Rho
GEF may be sufficient for activation.
Example 7. Effects of domains of p 11 S and Gam on the p 115 nucleotide
exchange activi~.
The theory that the RGS domain of p115 Rho GEF is normally autoinhibitory and
that binding to Ga,3 relieves this inhibition was examined by comparing the
effects of full-
length Rho-GEF versus truncated Rho-GEF on Rho exchange activity.
Preparation of p115 proteins was as described in Example 1 above and as
described
in Hart et al., J. Biol. Chem., 271, 25452-25458 ( 1996). The assays shown in
Figure 8,
Panels B and C were performed as described in Example 5 above. AMF was the
activating
agent.
The results of these experiments showed that truncated p 115 Rho GEF lacking
the
RGS domain demonstrates consistently elevated Rho exchange activity when
compared with
equal concentrations of the full-length protein (Fig. 8, Panel A).
Additionally, addition of
the isolated RGS domain (as a GST fusion protein) resulted in abrogation of
Ga,3-
stimulated p115 Rho GEF activity (Fig. 8, Panel B). These data do not preclude
additional
Ga,3-binding sites on p115 Rho GEF, although they do suggest a primary mode of
action
via the RGS domain.
The inability of the Ga,2 subunit to activate p115 Rho GEF was pu2zling in
light of
the fact that p115 Rho GEF is capable of activating the GTPase of both Ga,2
and Ga,3.
Therefore, an experiment was conducted in which Ga,2 was added to a Ga,3-
stimulated
p115 Rho GEF assay (Fig. 8, Panel C). The results showed that Ga,2 was able to
inhibit the
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WO 99/47557 PCT/US99/06051
coupling of Ga,3 with p115 Rho GEF. This data is consistent with a model in
which Ga,2
competes with Ga,3 for binding to the RGS domain of pl 15 Rho GEF. However,
binding
of Ga,2 to p115 Rho GEF is clearly not sufficient to stimulate Rho exchange
activity. These
results suggest that either the interaction of Ga,2 with the RGS domain of
p115 Rho GEF is
quite different from that of Ga,~ or that there may be an additional site of
interaction
between Ga, 3 and p 115 Rho GEF.
For other aspects of the nucleic acids, polypeptides, antibodies, etc.,
reference is
made to standard textbooks of molecular biology, protein science, and
immunology. See,
e.g., Davis et al. ( 1986), Basic Methods in Molecular Biology, Elsevir
Sciences Publishing,
Inc., New York; Hames et al. ( 1985), Nucleic Acid Hybridization, IL Press,
Molecular
Cloning, Sambrook et al.; Current Protocols in Molecular Biology, Edited by
F.M. Ausubel
et al., John Wiley & Sons, Inc; Current Protocols in Human Genetics, Edited by
Nicholas C.
Dracopoli et al., John Wiley & Sons, Inc.; Current Protocols in Protein
Science; Edited by
John E. Coligan et al., John Wiley & Sons, Inc.; Current Protocols in
Immunology; Edited
by John E. Coligan et al., John Wiley & Sons, Inc. The entire disclosure of
all patent
applications, patents, and publications cited herein are hereby incorporated
by reference.
From the foregoing description, on skilled in the art can easily ascertain the
essential
characteristics of this invention, and without departing from the spirit and
scope thereof, can
make various changes and modifications of the invention to adapt it to various
usages and
conditions.
